Hematopoietic stem cell transplantation (HSCT) would benefit from assays to ensure that the processed cells to be transplanted into a patient will home to the designated target organ, engraft or seed in that organ, initiate proliferation, and finally, reconstitute or repopulate the organ or tissue. Regardless of the cell source (bone marrow, mobilized peripheral blood, umbilical cord blood) and the type of transplantation performed (autologous or allogeneic), it is desirable to ensure the quality of the stem cells transplanted because, patients receiving the stem cell infusion have generally had their hematopoietic system partially or totally ablated by radiation and cytotoxic drugs and are at increased risk of dying if the transplanted cells do not engraft and repopulate their hematopoietic system. The first human autologous bone marrow transplantation (BMT) was performed by Kurnick et al. in 1958. Kurnick and Robinson, Colony growth of human peripheral white blood cells in vitro, Blood 37: 136 (1971). No assays to determine the quality of human transplanted cells with respect to their growth and engraftment potential were available until Pike and Robinson in 1971 applied the in vitro colony forming assays, first published in 1966, to human cells. Pike and Robinson, Human bone marrow colony growth in agar-gel, J Cell Physiol, 76: 77-84 (1970). Bradley and Metcalf, The growth of mouse bone marrow cells in vitro, Aust J Exp Biol Med, 44: 287 (1966).
The colony forming cell (CFC) assay (CFCA), first published in 1966 is a functional assay to detect morphologically unidentifiable stem and progenitor cells of the blood-forming (hematopoietic) tissue, because they are so few in number. The assay allows these cells to be stimulated in the presence of growth factors and cytokines and to undergo proliferation and division so that the daughter cells can differentiate into morphologically recognizable mature cells. This functional ability is detected by allowing the cells to grow in an immobilizing, semi-solid medium such as methylcellulose. As the cells divide, they remain in place and form a colony of cells that can be identified morphologically under an inverted microscope. Thus, even though the original cells that produce a colony cannot be identified morphologically, their functional ability to produce a colony can be detected and, therefore, by inference, their presence can be detected.
Despite the fact that the CFCA has been available for over 40 years, the technique has undergone few changes. The ability to detect multiple cell populations, however, even from the lymphopoietic system, has been established. The incorporation of recombinant growth factors and cytokines was introduced in the 1980s and 1990s as was the ability to culture the cells under low serum or serum-free conditions. Even with these additions, the assay has remained highly subjective due to the requirement to manually enumerate both the number and types of colonies produced. The subjectivity of the assay also means that the assay is extremely difficult to validate between different laboratories or even within a single laboratory due to the wide variation in what different individuals consider to be one type of colony versus another. Since there is no external parameter, such as a biochemical entity or process, to which the results can be compared, the CFCA assay has never been standardized. Regardless of the application for which the CFCA is used, there is no ability to compare the results of experiments or studies either within or between laboratories. As a result, if the CFCA is used as an end-point assay to control a particular procedure or process, for example, during the production of a stem cell product for transplantation into a patient, it is impossible for laboratories around the world to compare their results because of the non-standardized manner in which the colonies are manually enumerated. The result is that regulatory agencies have never been able to define specific criteria by which a procedure or process using the colony forming cell assay should be performed or the applicable range of results that should be acceptable.
The cell processing laboratory (CPL) is responsible for a quality product that is directly related to the success of the stem cell transplant. To this end, standards to maintain and enhance the quality and safety of the transplantation process through inspection and accreditation have been controlled by two groups in the United States, namely the American Association of Blood Banks (AABB) and Foundation for the Accreditation of Cellular Therapy (FACT), and in Europe by the Joint Accreditation Committee of ISCT-Europe and EBMT (JACIE). The U.S. Food and Drug Administration (FDA) has provided guidelines, especially since the implementation of gene therapy and ex vivo hematopoietic stem cell expansion protocols.
However, standards both the U.S. and Europe for ensuring that sufficient numbers of viable stem cells exhibiting proliferative or growth potential are distinctly lacking in information. There are two primary reasons for this. First, the absence of standardized, robust, and non-subjective assays, and second, a lack of consensus regarding the procedure or procedures to be used. For example, the JACIE standards state in Section D4.270, “For products undergoing manipulation that alters the final cell population, a relevant and validated assay, where available, should be employed for evaluation of the target cell population before and after the processing procedure(s)”. The Joint Accreditation Committee of ISCT-Europe and EBMT, Standards for hematopoietic progenitor cell collection, processing and transplantation (2003). Many transplant centers and umbilical cord blood storage facilities routinely perform colony-forming assays for quality control purposes and clinical monitoring in a stem cell transplantation setting. However, their use has been called into question. In an article by Henon et al. in 2001, the authors state, “Determination of the graft content in CFU-GM was the only one available until the end of the eighties. But, for technical reasons, and also because it does not actually evaluate the self-renewal potential of the cell products reinfused, it has now been commonly replaced by the determination of CD34+ cell amounts, which are known to contain the pluripotent hematopoietic stem cells.” Henon, et al., Importance of CD34+ cell subsets in autologous PBSC transplantation: The mulhouse experience using CD34+, J Biol Regul. Homeost. Agents, 15: 62-67 (2001).
Despite the availability of in vitro assays to detect stem cells with different degrees of “sternness” or primitiveness and, therefore, different degrees of self-renewal potential, the colony-forming assays suffer from many drawbacks. The assays are time-consuming to perform and require a high degree of technical expertise to manually enumerate and differentiate colonies. The assay is highly subjective and there is a lack of standardization in procedure, performance, and colony enumeration. From a scientific viewpoint, the most important parameter to ascertain is not whether the cells can differentiate but whether they can proliferate, since once proliferation is underway, differentiation invariably follows. Therefore, assessing the differentiation capability of cells, as detected in the colony-forming assay, is secondary to their ability to initiate and sustain proliferation.